WO2010137722A1

WO2010137722A1 - Method for screening of regenerative medicine

Info

Publication number: WO2010137722A1
Application number: PCT/JP2010/059169
Authority: WO
Inventors: 細谷昌樹; 國貞祐哉; 庄司昌伸
Original assignee: 武田薬品工業株式会社
Priority date: 2009-05-26
Filing date: 2010-05-25
Publication date: 2010-12-02
Also published as: EP2436775A1; EP2436775A4; JPWO2010137722A1; US20120142005A1

Abstract

A method for screening a substance capable of regulating the regeneration, proliferation or differentiation of a cell or organ, which comprises the steps of: (1) forming an embryonoid body from a cell having a regenerative, proliferative or differentiative capability; (2) treating the embryonoid body produced in step (1) with a digestive enzyme to prepare single cells of the embryonoid body; (3) seeding the cells prepared in step (2) onto an adhesive plate, adding a candidate substance to the plate, and carrying out the adhesive culture of the cells on the plate; (4) quantitatively analyzing the expression amounts of at least two of genes involved in the regeneration, proliferation or differentiation of cells simultaneously after the adhesive culture of step (3); and (5) evaluating the influence of the candidate substance on the regeneration, proliferation or differentiation of cells based on the results of the quantitative analysis obtained in step (4).

Description

Regenerative medicine screening method

The present invention relates to a screening method for regenerative medicine.

Regeneration is a phenomenon in which cells / tissues lacking in a living body are repaired by proliferation and differentiation of stem cells and the like. The constant renewal of cells that supply new cells in place of cells that have reached the end of their lives, such as skin and gastrointestinal epithelium, is called physiologic regeneration and replenishes cells and tissues that are rapidly lost due to damage or disease. What is restored is called pathological regeneration. Regeneration is an indispensable phenomenon for multicellular organisms to survive. However, in higher animals such as humans, there is a limit to the natural regenerative power, resulting in severe or extensive damage beyond this. Organs and tissues that do not heal will cause a life-sustaining crisis for the whole individual. Treatment of organ failure such as kidney, liver, and heart is already established for organ failure, but there are problems such as securing donors and immunocompatibility. There was a limit to the number of patients who could benefit from treatment. Regenerative medicine has recently attracted attention as a means of overcoming such problems of organ transplantation. This is to develop a technology for controlling the ability of a living body with respect to the generation and regeneration of tissues, and to reconstruct tissues and regenerate organs using cells collected from themselves or others. Already used in clinical practice are bone marrow transplantation, skin transplantation for burns, and islet transplantation in diabetes, which are performed in many diseases such as leukemia. In addition, neural stem cell transplantation for Parkinson's disease, bone marrow cell transplantation for myocardial infarction, and Schwann cell transplantation for spinal cord injury are also expected to be clinically applied. In addition, as for those utilizing growth differentiation factors, wound healing promotion using fibroblast growth factor (FGF), treatment of anemia with erythropoietin has already been used in the clinical field, and it depends on hepatocyte growth factor (HGF) or its gene. Revascularization is also expected to be put into practical use as a medicine.
The following techniques are known as techniques related to screening for regenerative medicine.
In WO2006-6722 (Patent Document 1), a gene involved in cell regeneration, proliferation or differentiation is analyzed by collectively analyzing the expression levels of a plurality of genes in cells having regeneration / differentiation / proliferation ability. A screening method for a gene involved in cell regeneration / proliferation / differentiation, which includes a specifying step, is disclosed.
Desbordes et al. Reported an example of screening a wide range of compounds involved in differentiation and self-replication (screening for compounds that fluctuate the expression of undifferentiation marker Oct4 in hES cells and identifying 14 compounds from 2880 compounds) ( Non-patent document 1).
Hahn et al. Have reported a screening method for a therapeutic agent for brain tumor focusing on HDAC inhibitors and the like (Non-patent Document 2).
Have reported a screening method using Multiplex RT-PCR (384-well plate), etc. (Non-patent Document 3).

WO2006-6722

Conventionally, the types of cells and organs that can be artificially prepared using stem cells (embryonic stem cells, somatic stem cells, etc.) and progenitor cells are limited, and there are many things such as pancreatic β cells, kidneys, and digestive tract Has not been able to produce cells and tissues that can be clinically applied. Even if the cells are useful to some extent in regenerative medicine, there are many cells that have not reached a level sufficient to completely compensate for the lost function. Technology to control cell differentiation more efficiently than before in order to be able to provide regenerative medicine to cells and tissues that cannot be prepared or have low efficiency despite the high clinical demand. is required. In addition, by applying a new mechanism that regulates cell differentiation that will become apparent in the course of technological development, a new screening system for drug candidate substances that promotes tissue reorganization and organ regeneration in vivo and externally will be established. Is possible. In particular, if a drug that activates and promotes regeneration of stem cells present in the living body can be developed, the burden on patients such as surgery and use of immunosuppressants can be eliminated. Furthermore, if all cells and tissues of humans or higher animals can be prepared efficiently and in large quantities, they can be applied to screening of drug candidate substances, evaluation of drug efficacy, safety tests, etc. in conventional drug discovery processes.
There are still many unclear points in the differentiation control process of cells, and there is a limit to regenerative differentiation control using known growth differentiation factors, cytokines, signal transduction system inhibitors and activators. In fact, the types of cells, tissues, and organs that can be regenerated in vitro or in vivo using existing differentiation regulators are very limited. In addition, screening using stem cells committed to a specific cell lineage yields substances that regulate the differentiation process into specific cells, but the substances obtained by such screening also act on other cells. In order to verify the possibility of having a difference, verification in another differentiation system is required. Furthermore, it is desirable to use stem cells obtained from human tissues to screen for substances that regulate the regenerative differentiation of human cells, but in terms of obtaining materials and preparing large numbers of cells for screening, etc. The stem cells that can be used are limited to bone marrow and adipose-derived mesenchymal stem cells and fetal-derived neural stem cells.
In this way, the living body has an endogenous mechanism that regulates regenerative differentiation, and it is possible to artificially regulate cell differentiation and proliferation using this mechanism. However, since there are still many unexplained regulatory mechanisms of actual regenerative differentiation, there has been a limit to the methods of allowing differentiation / growth factors to act on cells as conventionally known. In addition, since there are a wide variety of mechanisms related to regenerative differentiation, in order to find candidate substances for regenerative differentiation regulators or tools that efficiently regulate regenerative differentiation, the invention of a method for investigating its action widely and quantitatively has been invented. It was necessary.

As a result of intensive studies, the present inventors induced differentiation in multiple directions by forming embryoid bodies from Embryonic Stem (ES) cells, and dispersed embryoid bodies into single cells by trypsin treatment. Then, the compound to be evaluated was added and adhesion culture was performed, and a screening method for a regenerative medicine comprising a process for examining expression variation of two or more genes was found. Furthermore, as a selection criterion for genes used in evaluating gene expression variation due to candidate compounds, (i) increase or decrease in the differentiation process, (ii) expression can be detected as a significant value even without compound treatment (control), (Iii) The present inventors have found that it is useful to select as an index a gene (known) whose expression has been confirmed to vary in a pilot test using existing growth / differentiation factors. In general, there is a common mechanism in vertebrates for regenerative differentiation. Therefore, the knowledge obtained by using multipotent cells such as mouse ES cells, which are easy to prepare, can be used to regulate regenerative differentiation of human tissue stem cells. It has been found that it can be widely applied, and that there is a common regeneration differentiation mechanism among various stem cells, and that a substance that acts on certain stem cells can also act on other stem cells. As a result of further studies based on these findings, the present inventors have completed the present invention.
That is, the present invention provides the following screening method (hereinafter sometimes referred to as “the screening method of the present invention”).
[1] The following steps (1) to (5):
(1) a step of forming an embryoid body from cells having regeneration, proliferation or differentiation ability;
(2) A step of treating the embryoid body obtained in step (1) with a digestive enzyme to form a single cell state,
(3) A step of seeding the cells obtained in step (2) on an adhesive plate, adding a candidate substance to the plate, and subjecting the cells to adhesion culture,
(4) After the adhesion culture in step (3), a step of collectively analyzing the expression levels of two or more types of genes involved in cell regeneration, proliferation or differentiation, and (5) quantitative analysis in step (4) A method for screening a substance that regulates the regeneration, proliferation or differentiation of cells or organs, comprising the step of evaluating the influence of a candidate substance on the regeneration, proliferation or differentiation of cells based on the results of.
[2] The method of the above-mentioned [1], wherein the cells in the step (1) are selected from human and warm-blooded animal embryonic stem cells and iPS (Induced Pluripotent stem) cells.
[3] The method according to [1] or [2] above, wherein the period of cell culture performed to form embryoid bodies in the step (1) is 3 to 6 days.
[4] Cells obtained by inducing differentiation of human and warm-blooded animal embryonic stem cells, iPS (induced pluripotent stem) cells and those cells, which are targets of substances that regulate regeneration, proliferation or differentiation, and Tissue stem cells (mesenchymal stem cells, hematopoietic stem cells, myoblasts, neural stem cells, osteoblasts, chondroblasts, hemangioblasts, progenitor cells or stem cells present in the body) existing in living tissue or in The method according to any one of [1] to [3] above, which is selected from the group consisting of cells cultured in vitro.
[5] Any of the above [1] to [4], wherein the substance that regulates regeneration, growth or differentiation is a synthetic compound, natural product, protein, peptide, lipid, amine, amino acid, sugar, nucleic acid, or ion The method described in 1.
[6] Substances that regulate regeneration, proliferation or differentiation are receptor agonists and antagonists, biosynthetic pathway inhibitors, protein-protein interaction inhibitors, enzyme inhibitors and substrates, coenzymes, signal transduction system inhibitors and Activators, channel inhibitors and modulators, vitamins, antioxidants, apoptosis inhibitors and promoters, antiviral agents, surfactants, antisense oligonucleotides, siRNA, antibiotics, compounds synthesized by combinatorial chemistry methods, And the method according to any one of [1] to [4] above, which is selected from the group consisting of synthetic intermediates thereof.
[7] Cells or organs targeted by substances that regulate regeneration, proliferation or differentiation are splenocytes, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelium Cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes, megakaryocytes ), Synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, stem cells, blood cells, brain, brain parts (Olfactory bulb, cephalic nucleus, basal ganglia, hippocampus, thalamus, hypothalamus, hypothalamic nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, substantia nigra ), Spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad Thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (large and small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cell, prostate, testicle, testis, ovary, placenta, The method according to any one of [1] to [6] above, which is selected from the group consisting of uterus, bone, joint, and skeletal muscle.
[8] Substances that regulate regeneration, proliferation or differentiation are central diseases (Alzheimer's disease, Parkinson's disease, ischemic neuropathy), inflammatory diseases (allergic diseases, asthma, rheumatism, osteoarthritis), circulatory organs Disease (heart failure, cardiac hypertrophy, angina, arteriosclerosis), cancer (non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer), diabetes, Immune system disease (autoimmune disease, atopic dermatitis, allergic disease, immunodeficiency, asthma, rheumatoid arthritis, psoriasis, arteriosclerosis, diabetes, Alzheimer's disease), liver / gallbladder disease (cirrhosis, hepatitis, liver failure) , Cholestasis), digestive disorders (ulcers, enteritis, dyspepsia, irritable colitis, ulcerative colitis, diarrhea, ileus), burns, fractures, and alopecia It is a preventive / therapeutic The method according to any one of [1] to [7].
[9] The method according to any one of [1] to [8], wherein the digestive enzyme in the step (2) is trypsin.
[10] The method according to any one of [1] to [9], wherein the adhesive plate in the step (3) is a plate having a plurality of holes coated with gelatin.
[11] The method according to any one of [1] to [10] above, wherein Multiplex RT-PCR is used when quantitatively analyzing the expression levels of two or more genes in the step (4).
[12] A gene involved in cell regeneration, proliferation or differentiation in the step (4) is:
(A) Nanog, Oct3 / 4, Sox2, Klf4, Akp2, which are undifferentiated markers
(B) Fgf5, a primitive ectoderm marker,
(C) Brachyury, Snail1, which are primitive streak markers,
(D) Cdx2, Bmp4, which are trophectoderm marker
(E) Neural markers Tubb3, Nefh, Nestin, p75NTR,
(F) Actc1, a myocardial marker,
(G) Acta2, Cnn1, which are smooth muscle markers,
(H) Tie2, which is an endothelial cell marker,
(I) Flk1, which is a mesoderm marker,
(J) Cxcr4 which is a mesoderm and endoderm marker,
(K) Gata4, Laminin B1, which are extraembryonic endoderm markers,
(L) Acta1, Tpm1, which are skeletal muscle markers
(M) Opn which is an osteoblast marker,
The method according to any one of [1] to [11] above, which is selected from the group consisting of (N) c-kit which is a hematopoietic stem cell marker and (O) Sox9 which is a chondrocyte marker.

It is the table | surface which showed the arrangement | sequence of the primer and probe which were used for the measurement of the expression level of each gene. It is the table | surface which showed 52 compounds which raised the expression level of 1 or more gene more than 2 times compared with the control of DMSO addition as a result of the screening using LOPAC ¹²⁸⁰ (Sigma). The relative expression level of each gene when each compound is added is shown. The genes marked with * indicate 8 genes measured by screening. When it rises 2 times or more compared to the control to which DMSO was added, it is colored blue, and when it falls to 0.5 times or less, it is colored red. It is the table | surface which classified 52 compounds selected by the screening based on the pharmacological action. (A) It is a table | surface which shows the expression level of Adiponectin when 52 compounds shown in FIG. 2 were respectively added and the adhesion | cultivation culture | cultivation was carried out for 5 days simultaneously with induction | guidance | derivation of fat differentiation from a human mesenchymal stem cell. The values in the table indicate relative expression levels based on DMSO addition control. The case where it rose 1.3 times or more compared with the control which added DMSO is colored in blue, and the case where it falls below 0.5 times is colored in red. (B) It is a table | surface which shows the expression level of Alpl when each of the 52 compounds shown in FIG. 2 was added at the same time it induced | guided | derived bone differentiation from a human mesenchymal stem cell, and it culture | cultivated for 8 days. The values in the table indicate relative expression levels based on DMSO addition control. When it rises 2 times or more compared to the control to which DMSO was added, it is colored blue, and when it falls to 0.5 times or less, it is colored red. It is a figure which shows the expression level of an early differentiation marker when an embryoid body is formed by carrying out suspension culture using a 96-well spheroid plate. The value in the figure shows the relative expression level based on the expression level in undifferentiated ES cells on day 0 of culture. It is a figure which shows the expression level of an early differentiation marker in each stage of an undifferentiated ES cell (ES), an embryoid body (the suspension culture 4th day, EB), and adhesion culture | cultivation 1st-4th day. The value in the figure shows the relative expression level based on the expression level in undifferentiated ES cells on day 0 of culture. It is the figure which showed the outline of the screening method of this invention. In the screening method of the present invention, the expression of each differentiation marker is carried out by forming a embryoid body by culturing in suspension culture for 4 days, making it into a single cell state with a trypsin-EDTA solution, adding a compound, and further performing adhesion culture for 4 days. The procedure is to check the amount. It is a figure which shows the result of having added the compound in a figure according to the method shown in FIG. 7, and having measured the GAPDH expression level and the amount of ATP when carrying out adhesion culture for 4 days. The values in the figure show relative values based on DMSO addition control. It is a figure which shows the expression level of various genes when the compound in a figure is added according to the method shown in FIG. 7, and adhesion culture is carried out for 4 days. The value in the figure shows the relative expression level based on the control of DMSO addition. It is a figure which shows the result of having measured the amount of ATP when adding the compound in a figure according to the method shown in FIG. 7, and carrying out adhesion culture for 4 days. The values in the figure show relative values based on DMSO addition control. It is a figure of the result of having clustered the expression profile data of 28 genes based on correlation distance about 32 compounds among the compounds selected by screening. Compound 1-3 is a dopamine receptor antagonist, compounds 4 and 5 are p38 MAPK inhibitors, compound 6-9 is a corticosteroid, and compound 10-13 is a retinoic acid receptor agonist. (A) It is a figure which shows the expression level of Adiponectin and PPAR (gamma) when the compound in a figure is added simultaneously from the induction | guidance | derivation of fat differentiation from a human mesenchymal stem cell, and adhesion culture is carried out for 5 days. The value in the figure shows the relative expression level based on the control of DMSO addition. (B) It is a figure which shows the result of having dye | stained the lipid droplet using the oil red 0 solution, after adding the compound in a figure simultaneously with inducing | guiding | deriving fat differentiation from a human mesenchymal stem cell, and carrying out adhesion culture for 5 days. (C) It is a figure which shows the result of having measured the amount of adiponectin contained in a culture solution, after inducing fat differentiation from a human mesenchymal stem cell, adding the compound in a figure, and carrying out adhesion culture for 8 days. (A) It is a figure which shows the expression level of Alpl, Runx2, Col1a1, and PTHR when the compound in a figure is added simultaneously with induction | guidance | derivation of bone differentiation from a human mesenchymal stem cell, and adhesion culture is carried out for 8 days. The value in the figure shows the relative expression level based on the control of DMSO addition. (B) It is a figure which shows the result of having performed the alkaline phosphatase dyeing | staining after adding the compound in a figure and inducing | adhering culture | cultivation for 8 days simultaneously with inducing | generating bone differentiation from a human mesenchymal stem cell. (C) It is a figure which shows the result of having measured the amount of calcium accumulate | stored in the cell after adding the compound in a figure at the same time it induces bone differentiation from a human mesenchymal stem cell, and carrying out adhesion culture for 15 days. The values in the figure show relative values based on DMSO addition control.

1. According to one embodiment of the present invention, a screening method for a substance that regulates the regeneration, proliferation, or differentiation of a cell or organ (hereinafter referred to as “the present invention”). Is sometimes referred to as a screening method.). This method typically includes the following steps:
(1) A step of forming an embryoid body from cells having regeneration, proliferation or differentiation ability;
(2) A step of treating the embryoid body obtained in step (1) with a digestive enzyme to form a single cell;
(3) A step of seeding the cells obtained in step (2) on an adhesive plate, adding a candidate substance to the plate, and subjecting the cells to adhesion culture;
(4) After the adhesion culture in step (3), a step of collectively analyzing the expression levels of two or more types of genes involved in cell regeneration, proliferation or differentiation; and (5) quantitative analysis in step (4) The process of evaluating the influence of a candidate substance on cell regeneration, proliferation or differentiation based on the results of.
In the screening method of the present invention, first, in step (1), an embryoid body (EB) is formed from cells having regenerative, proliferative, or differentiation ability (eg, ES cells), so that it can be applied in multiple directions. Induces differentiation.
In the present specification, “embryoid body” is used in the meaning commonly used in the art, and refers to an embryonic form formed when pluripotent stem cells such as ES cells and iPS cells are differentiated in suspension culture. It means a cell cluster composed of stem cells or progenitor cells of various tissues.
In the screening method of the present invention, preferred examples of cells having the ability to regenerate, proliferate or differentiate used in the formation of the embryoid body in step (1) include embryonic properties of humans and other warm-blooded animals (eg, mice). Examples include stem cells (ES cells) and iPS (induced pluripotent stem) cells. In order to form an embryoid body, typically, ES cells such as mice are suspended in a medium containing serum or a serum replacement, and suspended in culture at 37 ° C. under 5% CO ₂ for 1 to 10 days. Particularly preferred examples of cells having the ability to regenerate, proliferate or differentiate used when forming the embryoid body in the step (1) include embryonic stem cells (ES cells) of humans and other warm-blooded animals (eg, mice). Can be mentioned.
The cell culture methods can be classified into adhesion culture and suspension culture according to the form of cells in the cell culture. Adhesive culture is a method of growing cultured cells by attaching them to a culture vessel, and suspension culture is a method of growing cultured cells in a floating state in a medium.
In the screening method of the present invention, cell culture performed to form embryoid bodies is typically performed in suspension culture. The culture period is not particularly limited as long as it is a period until an embryoid body is formed, but it is typically 1 to 6 days. The culture period is preferably 3 to 6 days. The culture period is more preferably 4 days.
For example, when an embryoid body is formed, a homogeneous embryoid body can be formed by using a non-adhesive multi-well plate, and a stable result can be obtained.
Next, in the step (2), a digestive enzyme is added to the embryoid body obtained in the step (1) to separate the cells into a single cell state. Examples of digestive enzymes that can be used include trypsin, collagenase, papain, dispase, and accutase (trade name). Of these, trypsin is preferable, and typically a trypsin-EDTA solution (eg, 0.25% trypsin-1 mM EDTA) added with EDTA to chelate Ca ²⁺ or Mg ²⁺ , which is an inhibitor of the action of digestive enzymes. ).
By preparing a homogeneous cell population derived from embryoid bodies in steps (1) and (2), it becomes possible to obtain stable results in screening.
In the step (3), the cells obtained in the step (2) are seeded on an adhesive plate, and a candidate substance is added to the plate for adhesion culture. In the present specification, the “adhesive plate” refers to a plate that can be used for adhesion culture, and a cell adhesion promoting substance, such as fibronectin, on the surface so that cells can adhere, spread, and proliferate on the plate. , A plate coated with type I or type IV collagen, laminin, vitronectin, gelatin, matrigel (trade name), polylysine, polyornithine. A preferable example of the adhesive plate used in the screening method of the present invention is a plate having a plurality of holes coated with gelatin (eg, 96-well plate).
In this specification, “candidate substance” refers to a candidate substance for a substance that regulates the regeneration, proliferation, or differentiation of cells or organs, that is, a substance to be screened by the screening method of the present invention. , Proteins, peptides, lipids, amines, amino acids, sugars, nucleic acids, ions, and the like. “Candidate substances” include receptor agonists and antagonists, biosynthetic pathway inhibitors, protein-protein interaction inhibitors, enzyme inhibitors and substrates, coenzymes, signal transduction system inhibitors and activators, channel inhibition. Agents and modulators, vitamins, antioxidants, apoptosis inhibitors and promoters, surfactants, antisense oligonucleotides, siRNA, antibiotics, antiviral agents, compounds synthesized by combinatorial chemistry methods, etc. Synthetic intermediates are also included.
The screening method of the present invention does not perform embryoid body formation in that the compound (candidate substance) is evaluated after the embryoid body containing cells differentiated in multiple directions is dispersed by digestion with a digestive enzyme such as trypsin. It is clearly distinguished from the conventional screening system using ES cells in which compound evaluation is performed by monolayer culture in an undifferentiated state.
In the step (4), after the adhesion culture in the step (3), two or more types of expression levels among genes involved in cell regeneration, proliferation or differentiation are collectively analyzed quantitatively.
In the present specification, the “gene involved in cell regeneration, proliferation or differentiation” is a gene already known to play a certain role in cell regeneration, proliferation or differentiation, or cell regeneration, A gene whose expression is known to vary greatly in the process of proliferation or differentiation.
In particular, the expression of “genes involved in cell regeneration, proliferation or differentiation” used in the screening method of the present invention is (i) expression is increased or decreased during the differentiation process, and (ii) significant value even without compound treatment (control). (Iii) It is preferable that the gene is confirmed to vary in expression in a pilot test using existing growth / differentiation factors.
Preferred examples of genes involved in cell regeneration, proliferation or differentiation that can be used in the screening method of the present invention include:
(A) Nanog, Oct3 / 4, Sox2, Klf4, Akp2, which are undifferentiated markers
(B) Fgf5, a primitive ectoderm marker,
(C) Brachyury, Snail1, which are primitive streak markers,
(D) Cdx2, Bmp4, which are trophectoderm marker
(E) Neural markers Tubb3, Nefh, Nestin, p75NTR,
(F) Actc1, a myocardial marker,
(G) Acta2, Cnn1, which are smooth muscle markers,
(H) Tie2, which is an endothelial cell marker,
(I) Flk1, which is a mesoderm marker,
(J) Cxcr4 which is a mesoderm and endoderm marker,
(K) Gata4, Laminin B1, which are extraembryonic endoderm markers,
(L) Acta1, Tpm1, which are skeletal muscle markers
(M) Opn which is an osteoblast marker,
(N) c-kit, which is a hematopoietic stem cell marker, and (O) Sox9, which is a chondrocyte marker.
In the present specification, “quantitative analysis of the expression levels of two or more genes among genes” is not performed sequentially one by one when performing quantitative analysis of the expression levels of two or more genes. It means that two or more kinds of genes are quantitatively analyzed together by one treatment. Specifically, for example, the expression levels of two or more types of genes can be measured simultaneously using, for example, Multiplex RT-PCR that can simultaneously measure the expression levels of a plurality of genes. Note that Multiplex RT-PCR is an existing technique, but the setting of the measurement system is complicated, and there are very few examples used for high-throughput screening. In the screening method of the present invention, it is preferable to use Multiplex RT-PCR in order to increase the system throughput in gene expression analysis. According to the screening method of the present invention, a wide group of compounds involved in differentiation can be obtained, and the obtained candidate compound group can be used for induction of differentiation from other adult stem cells or embryonic stem cells.
When performing quantitative analysis of two or more expression levels in a gene, it is preferable to measure an index reflecting the number of living cells as an internal control. By correcting the expression level of each gene with the internal control, the influence of the compound on the increase or decrease in the number of cells can be eliminated. Examples of indices reflecting the number of living cells include Gapdh expression level, ATP content, protein content, and DNA content. As an index reflecting the number of living cells, Gapdh expression level is preferable. In addition, as a control for observing changes in the expression level of the gene, the same solvent as that used for dissolving the compound, such as DMSO, DMF, methanol, ethanol, physiological saline, and buffer solution, is used. The gene expression level is 1.2 times or more, preferably 1.4 times or more, more preferably 1.6 times or more, even more preferably compared to the case where a control for monitoring the change in gene expression level is added. A substance that can be changed (decreased or increased) by 1.8 times or more, most preferably 2 times or more can be selected as a substance that regulates cell, organ regeneration, proliferation, or differentiation.
A target cell can be efficiently differentiated by causing a substance that regulates the regeneration, proliferation, or differentiation of cells or organs found by the screening method of the present invention to act on a stem cell alone or in combination.
2. Use of the compound obtained by the screening method of the present invention The compound obtained by the screening method of the present invention can be used to act on cells or organs to regulate regeneration, proliferation or differentiation. Such target cells include, for example, human and warm-blooded animal embryonic stem cells, iPS (induced pluripotent stem) cells, cells obtained by inducing differentiation of these cells, tissue stem cells (bone marrow and fat, etc.) Mesenchymal stem cells, hematopoietic stem cells, myoblasts, neural stem cells, osteoblasts, chondroblasts, hemangioblasts, and other progenitor cells or stem cells existing in the living body) or in vitro Examples include cultured cells.
Examples of cells or organs to be regenerated, proliferated, or differentiated by the action of the compound obtained by the screening method of the present invention include spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, and Langerhans cells. , Epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils Eosinophils, monocytes, megakaryocytes), synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, stem cells, etc. Hematopoietic cells, or any tissue in which these cells are present, such as the brain, brain regions (eg, olfactory bulb, buccal nucleus, basal basal sphere, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, Total , Cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, substantia nigra), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin , Muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cell, prostate, testis, testis, ovary, placenta, uterus, bone, joint and skeleton There are streaks.
Preferable examples of cells or organs to be regenerated, proliferated, or differentiated by the action of the compound obtained by the screening method of the present invention include bone cells and adipocytes.
The compound obtained by the screening method of the present invention, that is, a substance that regulates the regeneration, proliferation or differentiation of cells or organs can be used for treatment of diseases. Examples of target diseases include central diseases (eg, Alzheimer's disease, Parkinson's disease, ischemic neuropathy), inflammatory diseases (eg, allergic diseases, asthma, rheumatism, osteoarthritis), cardiovascular diseases (eg, , Heart failure, cardiac hypertrophy, angina, arteriosclerosis), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer), diabetes , Immune system diseases (eg autoimmune disease, atopic dermatitis, allergic disease, immunodeficiency, asthma, rheumatoid arthritis, psoriasis, arteriosclerosis, diabetes, Alzheimer's disease), liver / gallbladder disease (eg cirrhosis, Hepatitis, liver failure, cholestasis, stones), digestive disorders (eg, ulcers, enteritis, digestive failure, irritable colitis, ulcerative colitis, diarrhea, ileus), burns, fractures, alopecia, etc. disease Injury, and the like.
The compound obtained by the screening method of the present invention has low toxicity and, if necessary, is formulated according to a method known per se, and is orally or parenterally administered to a mammal (eg, human). be able to. Here, the dose and frequency of administration of the compound may be appropriately selected according to the administration subject, target disease and the like.
The relationship between the sequence numbers and sequences shown in the sequence listing of the present application is as follows.
[SEQ ID NO: 1]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 2]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 3]
The base sequence of the probe used in Example 1 is shown.
[SEQ ID NO: 4]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 5]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 6]
The base sequence of the probe used in Example 1 is shown.
[SEQ ID NO: 7]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 8]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 9]
The base sequence of the probe used in Example 1 is shown.
[SEQ ID NO: 10]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 11]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 12]
The base sequence of the probe used in Example 1 is shown.
[SEQ ID NO: 13]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 14]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 15]
The base sequence of the probe used in Example 1 is shown.
[SEQ ID NO: 16]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 17]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 18]
The base sequence of the probe used in Example 1 is shown.
[SEQ ID NO: 19]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 20]
The base sequence of the primer used in Example 1 is shown.
[SEQ ID NO: 21]
The base sequence of the probe used in Example 1 is shown.
[SEQ ID NO: 22]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 23]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 24]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 25]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 26]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 27]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 28]
The base sequence of the primer used in Example 2 is shown.
[SEQ ID NO: 29]
The base sequence of the primer used in Example 2 is shown.
[SEQ ID NO: 30]
The base sequence of the probe used in Example 2 is shown.
[SEQ ID NO: 31]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 32]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 33]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 34]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 35]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 36]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 37]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 38]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 39]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 40]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 41]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 42]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 43]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 44]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 45]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 46]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 47]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 48]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 49]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 50]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 51]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 52]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 53]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 54]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 55]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 56]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 57]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 58]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 59]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 60]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 61]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 62]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 63]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 64]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 65]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 66]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 67]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 68]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 69]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 70]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 71]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 72]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 73]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 74]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 75]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 76]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 77]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 78]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 79]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 80]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 81]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 82]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 83]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 84]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 85]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 86]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 87]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 88]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 89]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 90]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 91]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 92]
The base sequence of the primer used in Example 5 is shown.
[SEQ ID NO: 93]
The base sequence of the probe used in Example 5 is shown.
[SEQ ID NO: 94]
The base sequence of the primer used in Example 6 is shown.
[SEQ ID NO: 95]
The base sequence of the primer used in Example 6 is shown.
[SEQ ID NO: 96]
The base sequence of the probe used in Example 6 is shown.
[SEQ ID NO: 97]
The base sequence of the primer used in Example 6 is shown.
[SEQ ID NO: 98]
The base sequence of the primer used in Example 6 is shown.
[SEQ ID NO: 99]
The base sequence of the probe used in Example 6 is shown.
[SEQ ID NO: 100]
The base sequence of the primer used in Example 6 is shown.
[SEQ ID NO: 101]
The base sequence of the primer used in Example 6 is shown.
[SEQ ID NO: 102]
The base sequence of the probe used in Example 6 is shown.
[SEQ ID NO: 103]
The base sequence of the primer used in Example 7 is shown.
[SEQ ID NO: 104]
The base sequence of the primer used in Example 7 is shown.
[SEQ ID NO: 105]
The base sequence of the probe used in Example 7 is shown.
[SEQ ID NO: 106]
The base sequence of the primer used in Example 7 is shown.
[SEQ ID NO: 107]
The base sequence of the primer used in Example 7 is shown.
[SEQ ID NO: 108]
The base sequence of the probe used in Example 7 is shown.
[SEQ ID NO: 109]
The base sequence of the primer used in Example 7 is shown.
[SEQ ID NO: 110]
The base sequence of the primer used in Example 7 is shown.
[SEQ ID NO: 111]
The base sequence of the probe used in Example 7 is shown.
[SEQ ID NO: 112]
The base sequence of the primer used in Example 7 is shown.
[SEQ ID NO: 113]
The base sequence of the primer used in Example 7 is shown.
[SEQ ID NO: 114]
The base sequence of the probe used in Example 7 is shown.
[SEQ ID NO: 115]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 116]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 117]
The base sequence of the probe used in Example 3 is shown.
[SEQ ID NO: 118]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 119]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 120]
The base sequence of the probe used in Example 3 is shown.
[SEQ ID NO: 121]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 122]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 123]
The base sequence of the probe used in Example 3 is shown.
[SEQ ID NO: 124]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 125]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 126]
The base sequence of the probe used in Example 3 is shown.
[SEQ ID NO: 127]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 128]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 129]
The base sequence of the probe used in Example 3 is shown.
[SEQ ID NO: 130]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 131]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 132]
The base sequence of the probe used in Example 3 is shown.
[SEQ ID NO: 133]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 134]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 135]
The base sequence of the probe used in Example 3 is shown.
[SEQ ID NO: 136]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 137]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 138]
The base sequence of the probe used in Example 3 is shown.
The following abbreviations used in the present specification are according to the examples currently commonly used in this technical field, and their meanings are as follows.
DMEM: Dulbecco's Modified Eagle Medium
MEM: Minimum Essential Medium
RT-PCR: Reverse Transcript Polymerase Chain Reaction
ATP: Adenosine triphosphate DMSO: Dimethyl Sulfoxide
EDTA: ethylenediaminetetraacetic acid BrdU: 5-bromo-2′-deoxyuridine
PBS: Phosphorate buffered sale
ELISA: Enzyme-Linked Immunosorbent Assay
EXAMPLES Hereinafter, although this invention is demonstrated more concretely using an Example, the scope of the present invention is not limited to such an example.

Example 1: Construction of a compound evaluation system (embryoid body formation)
D3 strain (American Type Culture Collection, CRL-1934) was used as ES cells. In order to maintain ES cells, mouse fibroblasts (MEF) (purchased from Millipore) whose cell growth was stopped by mitomycin C treatment were seeded on gelatin-coated plates and used as feeders. After seeding the ES cells on a feeder, 20% calf fetal serum (FBS, ES cell-qualified, Invitrogen), 100 μM 2-mercaptoethanol (Invitrogen), 1 × MEM non-essential amino acid solution (DMEM (Invitrogen)) Invitrogen), 1x antibiotic-anticolytic (Invitrogen), 1000 U / ml leukemia inhibitory factor (LIF, Chemicon) was added to the culture solution at 37 ° C, 5% CO ₂ Cultured under. The medium was changed every day, and the undifferentiated state was maintained by subculture every 3 days using a 0.25% trypsin-1 mM EDTA solution (Invitrogen, hereinafter referred to as “trypsin-EDTA solution”).
In order to stabilize and simplify the compound evaluation system, a large amount of undifferentiated cells from which feeder cells had been removed were stored frozen. First, the cells were made into a single cell state using a trypsin-EDTA solution, transferred to a gelatin-coated culture dish, and cultured at 37 ° C. for 90 minutes. The cells that did not adhere were recovered to remove mouse fibroblasts (MEF) as a feeder, and then cultured for another 3 days in another gelatin-coated culture dish. Thereafter, cells made into a single cell state again using trypsin-EDTA solution were placed in a gelatin-coated culture dish at 1 × 10 5. ⁵ cells / cm ² And the cells were further cultured for 24 hours. After detaching the cells with trypsin-EDTA solution, 2 × 10 ⁶ The suspension was suspended in a cell banker (Nippon Zenyaku Kogyo Co., Ltd.) at a concentration of cells / ml and stored frozen at -80 ° C. Subsequent experiments were performed using the cryopreserved cells.
In order to form embryoid bodies, the cryopreserved cells were lysed in a 37 ° C. constant temperature bath, and then seeded on a 96-well spheroid plate (Sumitomo Bakelite) at 1000 cells per well. 10% calf fetal serum (FBS, ES cell-qualified, Invitrogen), 2 mM GlutaMAX-1 (Invitrogen), 3 × 10 in DMEM medium ^-4 Suspension culture was performed for 1 to 6 days using a culture solution supplemented with M monothioglycerol (Sigma), 1 × MEM non-essential amino acid solution (Invitrogen), and 1 × penicillin-streptomycin (Sigma). Conventionally, by using a 96-well spheroid plate to form one embryoid body per well, floating culture is performed on a non-adhesive culture dish to form a heterogeneous embryoid body. Compared with this method, embryoid bodies with a uniform size were formed.
Changes in the expression of early differentiation markers during the process of embryoid body formation using this technique were examined. Embryoid bodies were collected over time, and total RNA was purified using RNeasy 96 or RNeasy mini kit (Qiagen). After synthesizing cDNA using PrimeScript RT reagent kit (Takara Bio Inc.), quantitative RT-PCR was performed to measure the expression levels of Brachyury, Flk1, Sox17, Sox1, Oct3 / 4, and Nanog genes. . The primer and probe sequences used for measuring each gene are shown in FIG.
FIG. 5 shows the results of analyzing the expression levels of Brachyury, Flk1, Sox17, Sox1, Oct3 / 4, and Nanog genes. From the 4th day to the 5th day of suspension culture, a transient increase in expression of Brachyury, a primitive streak marker, was observed. Furthermore, the expression of Flk1, which is a mesoderm marker, and Sox17, which is an endoderm marker, were also significantly increased from the 4th day to the 5th day of suspension culture. In addition, the expression of Sox1, which is an ectoderm marker, gradually increased during the culture period. On the other hand, the expression of Oct3 / 4 and Nanog, which are undifferentiated markers, gradually decreased as the suspension culture period became longer. From these results, it was confirmed that the embryoid body having a uniform size was formed by using the method described in this example, and that ES cells were cultured in suspension culture for 4 days or more to move the ES cells in the direction of 3 germ layers. It became clear that differentiation was induced.
Example 2: Construction of a compound evaluation system (transfer from embryoid body to adhesion culture)
In order to enable high-throughput compound evaluation, embryoid bodies were dispersed and adhesion culture was performed by the following method. According to the method described in Example 1, suspension culture was performed for 4 days to prepare embryoid bodies, and then treated with a trypsin-EDTA solution to obtain a single cell state. Cells in a single cell state were seeded in a gelatin-coated 96-well plate (Asahi Techno Glass) at 1000 cells per well, and adhesion culture was performed for 1 to 4 days. The culture solution used was the same as that used during embryoid body formation. The expression level of the initial differentiation marker in the adhesion culture for 1 to 4 days was measured by using the same method as in Example 1. The primer and probe sequences used for measuring each gene are shown in FIG.
FIG. 6 shows the results of analyzing the expression level of the early differentiation marker in the adhesion culture for 1 to 4 days. The expression levels of Flk1 and Brachyury expressed in mesoderm were significantly increased by adhesion culture. In addition, while the expression level of Sox1 as an ectoderm marker was decreased by adhesion culture, the expression level of Nestin as a neural progenitor cell marker was gradually increased. In addition, expression of Cdx2, which is a trophectoderm marker, was also significantly induced by adhesion culture. On the other hand, the expression of Foxa2 which is an endoderm marker was decreased by adhesion culture. From these results, it was clarified that differentiation into the mesodermal system, nervous system, and trophectoderm was induced even after the transfer from embryoid bodies to adhesion culture.
Based on the above results, a compound evaluation system using ES cells was set up. The outline of this evaluation system is shown in FIG. In this evaluation system, (1) embryoid bodies are formed by carrying out suspension culture for 4 days using a 96-well spheroid plate. (2) The embryoid bodies are treated with a trypsin-EDTA solution. After cell state, seed in gelatin-coated 96-well plate, (3) Add compound to be evaluated at the same time as seeding of cell, and perform adhesion culture for 4 days, (4) Expression of each differentiation marker at the end of culture It consists of the procedure of evaluating the effect of a compound on differentiation by measuring the amount. As differentiation markers to be measured, Nanog as an undifferentiated marker, Tub3 and Nefh as neuronal markers, Actc1 as a cardiac muscle marker, Acta2 as a smooth muscle marker, Tie2 as an endothelial cell marker, Flk1 as a mesodermal marker 7 A gene was selected. Furthermore, the Gapdh expression level was also measured as an internal control reflecting the number of living cells. In order to evaluate the influence of the compound on the number of living cells, the Gapdh expression level was also set as one item for evaluation. In order to avoid the influence of increase / decrease in the number of cells, the expression level of the other differentiation marker was used for the evaluation of the compound.
Example 3: Verification of a compound evaluation system using compounds involved in differentiation
In order to verify whether the screening method of the present invention is effective in detecting compounds involved in differentiation, the expression variation of differentiation markers when various compounds reported to be involved in differentiation were added was examined. . According to the protocol shown in Example 2, the compounds involved in differentiation were evaluated.
As a result, 1 μM 6-bromodirubin-3′oxime (BIO, GSK3β inhibitor), 10 μM PD169316 (p38 MAPK inhibitor), 50 nM retinoic acid, 3 μM dexamethasone, 0.5 μM BIX-01294 (G9a histone methyltransferase inhibitor), It was found that when 1 μM SB 431542 (ALK5 inhibitor) and 1 μM subheroylilidehydroacid (SAHA, HDAC inhibitor) were added, the expression level of one or more kinds of genes was increased more than twice compared with the addition of DMSO. Moreover, the pattern of increase / decrease in the expression of the eight types of genes by compound addition was different for each compound, and it was estimated that each compound had different effects on differentiation. Since the activities of various compounds involved in differentiation could be detected by the screening method of the present invention, it was confirmed that the screening method of the present invention is very useful in searching for a new differentiation regulator.
Example 4: Compound evaluation using LOPAC library
LOPAC, a commercially available compound library, using the screening method of the present invention ¹²⁸⁰ Screening of compounds involved in differentiation was carried out from (1280 compounds, Sigma). According to the protocol shown in Example 2, each compound was added so that the final concentration was 3 μM and the DMSO concentration as a solvent was 0.15%, and the compound evaluation was performed. In the screening, a Multiplex RT-PCR system capable of simultaneously measuring the expression levels of a plurality of genes was set in order to measure the expression levels of the eight types of genes shown above with high throughput. Eight types of gene expression are set 1 (Gapdh (fluorescent probe BODIPY), Acta2 (fluorescent probe VIC), set 2 (Actc1 (fluorescent probe BODIPY), Tubb3 (fluorescent probe VIC), Nanog (fluorescent probe FAM)), set 3 (Flk1 (fluorescent probe BODIPY), Nefh (fluorescent probe VIC), and Tie2 (fluorescent probe FAM)) were measured by multiple multiplex RT-PCR. Shown in
As a result of screening, the expression level of Gapdh was reduced to 5% or less for 56 compounds with respect to the control with DMSO addition, suggesting that these compounds are cytotoxic. These 56 compounds were excluded from the subsequent analysis. Among the other compounds, a compound having an expression level increased by a factor of 2 or more was selected in at least one gene as compared with the DMSO-added control. Furthermore, for the active compounds, the same experiment was conducted once again to examine reproducibility, and 52 compounds (4.1% of all compounds) whose reproducibility was finally confirmed were hit compounds (hereinafter referred to as “selection”). (Sometimes referred to as “compounds”) (FIG. 2). The pharmacological action of the hit compound is shown in FIG. The hit rate for each gene is 2.7% for Flk1, 0.2% for Actc1, 1.6% for Acta2, 0.6% for Tie2, 0.5% for Nefh, and 0.4% for Tubb3 , Nanog was 0.0% and Gapdh was 1.1%. From these results, it became clear that the activity of more compounds can be detected by measuring the expression of 8 types of genes simultaneously than when measuring the expression of only 1 type of gene. Further, the compounds selected as hit compounds are known to be involved in differentiation in other reports, and BIO, PD169316, retinoic acid, which were also recognized in the compound evaluation system described in this example. These compounds were included. These results indicate that the compound involved in differentiation was correctly selected in this screening method. The hit compounds contained 14 compounds that increase the Gapdh expression level. In order to examine the effect of these compounds on the number of viable cells, the amount of ATP in the well was measured using CellTiter-Glo luminescent cell viability kit (Promega). As a result, the amount of ATP in the well was increased by the addition of each 14 compounds. From these results, it was revealed that these 14 compounds have an action of increasing the number of living cells at least under the differentiation conditions.
Example 5: Effect of compounds selected from screening on expression of other differentiation markers
In order to analyze the effect of the selected compound on differentiation of ES cells in more detail, the expression level of differentiation markers other than the 8 genes used in the screening was also measured by the same method as described above. As differentiation markers, undifferentiation markers Oct3 / 4, Sox2, Klf4, Akp2, primitive ectoderm marker Fgf5, primitive streak markers Brachyury, Snail1, trophectoderm markers Cdx2, Bmp4, mesoderm and Cxcr4 which is an endoderm marker, Gata4 which is an extraembryonic endoderm marker, Laminin B1, Nestin which is a neuronal marker, p75NTR, Acta1 and Tpm1 which are skeletal muscle markers, Cnn1 which is a smooth muscle marker, Opn which is an osteoblast marker The expression levels of c-kit, a hematopoietic stem cell marker, and Sox9, a chondrocyte marker were measured. The primer and probe sequences used for measuring each gene are shown in FIG.
Of the selected compounds, 32 compounds were found to have an activity of increasing the expression of two or more types of genes more than twice (FIG. 2). In order to compare the gene expression patterns of these 32 kinds of compounds, clustering based on correlation distance was performed for each expression data.
The result is shown in FIG. As a result, it was clarified that compounds having the same pharmacological action and similar in compound structure show similar gene expression profiles. For example, thiothixene hydrochloride, perphenazine, cis- (Z) -fluxendix dihydrochloride, which are similar in structure to dopamine receptor antagonists, were classified into the same group. PD 169316 and SB 202190, both of which are p38 MAPK inhibitors and similar in structure, were also classified into the same group. Furthermore, corticosteroids hydrocortisone 21-hemisuccinate sodium, hydrocortisone, betamethasone, beclomethasone, retinoic acid receptor agonists TTNPB, 13-cis-retinoic acid, It was. As described above, by analyzing the expression levels of a plurality of differentiation markers using the compound evaluation system of this example, it was revealed that the compounds can be classified based on the effect on differentiation.
Example 6: Effect of compounds selected by screening on adipose differentiation from human mesenchymal stem cells
It was considered that the selected compound could control differentiation from adult stem cells as well as ES cells. Therefore, the effect of these selected compounds on adipogenesis from human mesenchymal stem cells was examined. Human mesenchymal stem cells were purchased from Lonza and medium supplemented with 10% calf fetal serum, 2 mM GlutaMAX-1, 1x penicillin-streptomucin (Sigma) (hereinafter “growth medium”) in low glucose DMEM (Invitrogen). The culture was performed using an adhesive culture. At the time of induction of adipose differentiation, cells were seeded in a 96-well plate so that the number of cells was 3000 per well, and then cultured overnight in a growth medium. Thereafter, the growth medium was added to a fat differentiation induction medium (high Golcose DMEM (Invitrogen)), 10% calf fetal serum, 2 mM GlutaMAX-1, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Wako Pure Chemical Industries), 10 μg / ml. Adipose differentiation was induced by exchanging with insulin (Wako Pure Chemicals), 1 μM dexamethasone (Wako Pure Chemicals), 200 μM indomethacin (Wako Pure Chemicals), and 1xpenicillin-streptomucin (Sigma)). At the same time as induction of adipose differentiation, each selected compound was added to a final concentration of 3 μM and cultured for 5 days. Five days later, the expression level of Adiponectin, which is an adipocyte marker, was measured using the same method as shown in Example 1. The primer and probe sequences used for measuring each gene are shown in FIG.
Among the selected compounds, ROCK inhibitor Y-27632, kinase inhibitor HA-100, and vanilloid receptor antagonist capsazepine were found as compounds that increase the expression level of Adiponectin (FIG. 12a). The action of these compounds to increase Adiponectin expression was equivalent to or better than that of PPARγ agonist Troglitazone (10 μM).
Furthermore, the expression level of PPARγ, which is a master regulator of adipose differentiation when these three compounds (Y-27632, HA-100, and capsazepine) were added, was also measured by the same method. As shown in FIG. 12a, it was confirmed that Y-27632 and HA-100 have an effect of increasing the PPARγ expression level in a concentration-dependent manner.
Next, the effect of these three compounds on lipid droplet formation was examined. 10 μM Y-27632, 10 μM HA-100, or 3 μM capsazepine was added to the adipose differentiation-inducing medium at the same time as induction of adipose differentiation, followed by adhesion culture for 5 days. Thereafter, each cell was fixed with 4% paraformaldehyde (Wako Pure Chemical Industries) for 30 minutes and then washed with PBS three times. Furthermore, after staining with 0.36% Oil Red 0 (Chemicon) for 50 minutes at room temperature, the cells were washed three times with distilled water to observe and photograph the cells. As a result, it was observed that the addition of Y-27632 and HA-100 increased the amount of lipid droplets compared to the DMSO addition control (FIG. 12b). On the other hand, capsazepine did not show significant activity to increase lipid droplet formation.
Furthermore, when these three compounds were added, the amount of adiponectin contained in each culture solution was measured. 10 μM Y-27632, 10 μM HA-100, 3 μM capsazepine were added simultaneously with the induction of adipogenesis, and adhesion culture was performed for 8 days. After culturing, the amount of adiponectin contained in the culture solution was measured using a human adipectin ELISA kit (R & D systems). As a result, it was revealed that the amount of adiponectin contained in the culture broth was significantly increased by adding Y-27632 and HA-100 (FIG. 12c). On the other hand, capsazepine did not have an effect of increasing the amount of adiponectin. From the above results, it was revealed that at least Y-27632 and HA-100 have an activity of promoting fat differentiation from human mesenchymal stem cells. On the other hand, among the selected compounds, there are 10 compounds that reduce the expression level of Adiponectin to half or less, suggesting that the selected compounds include compounds that suppress fat differentiation.
Example 7: Effect of compounds selected from screening on bone differentiation from human mesenchymal stem cells
Next, the influence of selected compounds on bone differentiation from human mesenchymal stem cells was examined. Human mesenchymal stem cells were maintained and cultured by the same method as in Example 6. Cells were seeded in a 96-well plate at 1000 cells per well and then cultured overnight in growth medium. Then, 10% calf fetal serum, 2 mM GlutaMAX-1, 10 mM β-glycerophosphate (Wako Pure Chemical), 0.05 mM ascorbic acid-2-phosphate (Wako Pure Chemical), 0.1 μM dexamethasone (Wako Pure Chemical) ) Bone differentiation was induced by replacing the medium with a medium supplemented with 1x penicillin-streptomycin (Sigma). At the same time as inducing bone differentiation, each of the selected compounds was added so that the final concentration was 3 μM, and adhesion culture was performed for 8 days. After completion of the culture, the expression level of Alkaline phosphatase (Alpl), which is an early osteoblast marker, was measured by the same method as in Example 1. The primer and probe sequences used for measuring each gene are shown in FIG.
As a result, among the selected compounds, GSK3β inhibitor 6-bromodirubin-3′oxime (BIO) and thymidine analog 5-Bromo-2′-deoxyuridine (BrdU) are compounds that significantly increase the expression level of Alpl. Found (Figure 13a).
The expression of other bone differentiation markers when BIO and BrdU were added was also examined. After BIO and BrdU were added and adherent culture was carried out for 8 days, runt-related transcription factor 2 (Runx2), collagen type I alpha 1 (Col1a1), and parathyroid hormone PT content amount were expressed in the same manner as in Example 1. It was measured. The primer and probe sequences used for measuring each gene are shown in FIG. As a result, it was revealed that the expression levels of Runx2, Col1a1, and PTHR were significantly increased by 3 μM BIO and 10 μM BrdU (FIG. 13a).
To further verify the effect of the compound on bone differentiation, alkaline phosphatase staining was performed. At the same time as inducing bone differentiation, 3 μM BIO and 10 μM BrdU were added, followed by adhesion culture for 8 days. After culturing, the cells were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries) for 30 minutes and then washed 3 times with PBS. Alkaline phosphatase kit III (Vector Laboratories) was used for alkaline phosphatase staining according to the attached protocol. As a result, it was revealed that the addition of BIO and BrdU increases the staining property of alkaline phosphatase, and these compounds increase the alkaline phosphatase activity (FIG. 13b).
Furthermore, the amount of calcium accumulated in the cells was examined. At the same time as inducing bone differentiation, 3 μM BIO and 10 μM BrdU were added, followed by adhesion culture for 15 days. After culturing, the cells were washed with PBS and then lysed in a lysate consisting of 10 mM Tris-HCl and 0.2% Triton X-100. The protein concentration was measured using BCA protein assay kit (Pierce) using a part of the lysate. Hydrochloric acid was added to the remaining lysate so that the final concentration was 0.5 N, and the mixture was shaken overnight. The amount of calcium dissolved in the solution containing 0.5N hydrochloric acid was measured using Calcium E-test Kit Wako (Wako Pure Chemical Industries). The amount of calcium corrected for the amount of protein was used for analysis. As a result, the amount of intracellular calcium was increased by the addition of BIO and BrdU (FIG. 13c). From these results, it became clear that BIO and BrdU have the activity of promoting bone differentiation from human mesenchymal stem cells. On the other hand, there are two compounds that reduce the Alpl expression level by half or less among the selected compounds, suggesting that the selected compounds include compounds that suppress bone differentiation.
Example 8: Correlation between Gapdh expression level and ATP level in the screening method of the present invention
It was examined whether the Gapdh expression level measured in the screening method of the present invention correlated with the number of living cells in the well. According to the method shown in Example 2, 1 μM 6-bromodirubin-3′oxime (BIO), 10 μM PD169316, 10 μM forskolin, 50 nM retinoic acid, 5 μM myoseverin, 3 μM dexamethasone, which is a compound known to be involved in differentiation, 0 After adding 5 μM BIX-01294 and adhesion culture for 4 days, the expression level of Gapdh was measured in the same manner as in Example 1. Simultaneously with the above measurement, the amount of ATP in the wells was also measured using CellTiter-Glo luminescent cell viability kit (Promega). The result is shown in FIG. When any compound was added, the Gapdh expression level correlated very well with the ATP level in the well. From these results, it was confirmed that the Gapdh expression level is useful as an index indicating the number of living cells in the well.
Example 9: Compound evaluation using compounds involved in differentiation
In Example 3, it was shown that when a compound known to be involved in differentiation was added, eight types of gene expression patterns fluctuated. The results of these experiments are shown in FIG. As shown in Example 3, it was found that when each compound was added, the expression level of one or more kinds of genes increased more than twice as compared with the case of adding DMSO. Moreover, the pattern of increase / decrease in the expression of the eight types of genes by compound addition was different for each compound, and it was estimated that each compound had different effects on differentiation. Since the activities of various compounds involved in differentiation could be detected by the screening method of the present invention, it was confirmed that the screening method of the present invention is very useful in searching for a new differentiation regulator.
Example 10: Effect of Compound with Increased Gapdh Expression on Viable Cell Count
In Example 4, 14 types of compounds that increase the expression level of Gapdh were found. After adding these 14 compounds, the result of having measured the amount of ATP in a well using CellTiter-Glo Luminescent cell viability kit (Promega) is shown in FIG. As described in Example 4, when these 14 compounds were added, an increase in the amount of ATP in the well was observed in all cases. These 14 compounds were found to have the effect of increasing the number of living cells under the conditions of the screening method of the present invention.
Example 11: Effect of compounds selected from screening on differentiation from human mesenchymal stem cells
Based on the technique shown in Example 6 and Example 7, the effect of the selected compound on fat differentiation or bone differentiation from human mesenchymal stem cells was examined. FIG. 4 summarizes the influence of the selected compound on the expression level of Adiponectin when inducing fat differentiation and on the expression level of Alpl in inducing bone differentiation. Y-27632, HA-100, and capsazepine were found as compounds that increase the expression level of Adiponectin 1.3 times or more as compared with the control when added during induction of adipogenesis. In addition, BIO and BrdU were found as compounds that increase the Alpl expression level by a factor of 2 or more as compared to the control when added during the induction of bone differentiation.

The screening method of the present invention is useful for screening drug candidates that activate stem cells and the like existing in a living body and promote regeneration. The pharmaceutical obtained by the screening method of the present invention can be used to eliminate the burden on patients such as surgery and use of immunosuppressive agents. Furthermore, since the screening method of the present invention can be used to efficiently and in large quantities prepare all cells and tissues of humans or higher animals, screening of drug candidates and drug efficacy in conventional drug discovery processes are possible. It can be applied to evaluation and safety testing.

Claims

The following steps (1) to (5):
(1) a step of forming an embryoid body from cells having regeneration, proliferation or differentiation ability;
(2) A step of treating the embryoid body obtained in step (1) with a digestive enzyme to form a single cell state,
(3) A step of seeding the cells obtained in step (2) on an adhesive plate, adding a candidate substance to the plate, and subjecting the cells to adhesion culture,
(4) After the adhesion culture in step (3), a step of collectively analyzing the expression levels of two or more types of genes involved in cell regeneration, proliferation or differentiation, and (5) quantitative analysis in step (4) A method for screening a substance that regulates the regeneration, proliferation or differentiation of cells or organs, comprising the step of evaluating the influence of a candidate substance on the regeneration, proliferation or differentiation of cells based on the results of.
The method according to claim 1, wherein the cells in the step (1) are selected from human and warm-blooded animal embryonic stem cells and iPS (induced primipotent stem) cells.
The method according to claim 1, wherein the period of cell culture performed to form embryoid bodies in the step (1) is 3 to 6 days.
Cells that are targets of substances that regulate regeneration, proliferation or differentiation are embryonic stem cells, iPS (induced primepotent stem) cells of humans and warm-blooded animals, cells obtained by inducing differentiation of these cells, and tissue stem cells. The method according to claim 1, wherein the method is selected from the group consisting of cells existing in a living tissue or cultured in vitro.
The method according to claim 1, wherein the substance that regulates regeneration, growth or differentiation is a synthetic compound, natural product, protein, peptide, lipid, amine, amino acid, sugar, nucleic acid, or ion.
Substances that regulate regeneration, proliferation or differentiation are receptor agonists and antagonists, biosynthetic pathway inhibitors, protein-protein interaction inhibitors, enzyme inhibitors and substrates, coenzymes, signal transduction system inhibitors and activators , Channel inhibitors and modulators, vitamins, antioxidants, apoptosis inhibitors and promoters, antiviral agents, surfactants, antisense oligonucleotides, siRNA, antibiotics, compounds synthesized by combinatorial chemistry methods, and their 2. The method of claim 1, wherein the method is selected from the group consisting of synthetic intermediates.
Cells or organs targeted by substances that regulate regeneration, proliferation or differentiation are spleen cells, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibers Blast cells, fiber cells, muscle cells, adipocytes, immune cells, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, Blood cells, brain, brain parts, spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract, blood vessel, heart, thymus, The method of claim 1, wherein the method is selected from the group consisting of spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testis, testis, ovary, placenta, uterus, bone, joint, and skeletal muscle.
Substances that regulate regeneration, proliferation or differentiation consist of central diseases, inflammatory diseases, cardiovascular diseases, cancer, diabetes, immune system diseases, liver / gallbladder diseases, digestive system diseases, burns, fractures, and alopecia The method according to claim 1, which is a prophylactic / therapeutic agent for a disease selected from the group.
The method according to claim 1, wherein the digestive enzyme in the step (2) is trypsin.
The method according to claim 1, wherein the adhesive plate in the step (3) is a plate having a plurality of holes coated with gelatin.
The method according to claim 1, wherein when the expression levels of two or more genes are collectively analyzed in the step (4), Multiplex RT-PCR is used.
Genes involved in cell regeneration, proliferation or differentiation in the step (4) are:
(A) Nanog, Oct3 / 4, Sox2, Klf4, Akp2, which are undifferentiated markers
(B) Fgf5, a primitive ectoderm marker,
(C) Brachyury, Snail1, which are primitive streak markers,
(D) Cdx2, Bmp4, which are trophectoderm marker
(E) Neural markers Tubb3, Nefh, Nestin, p75NTR,
(F) Actc1, a myocardial marker,
(G) Acta2, Cnn1, which are smooth muscle markers,
(H) Tie2, which is an endothelial cell marker,
(I) Flk1, which is a mesoderm marker,
(J) Cxcr4 which is a mesoderm and endoderm marker,
(K) Gata4, Laminin B1, which are extraembryonic endoderm markers,
(L) Acta1, Tpm1, which are skeletal muscle markers
(M) Opn which is an osteoblast marker,
The method according to claim 1, wherein the method is selected from the group consisting of (N) hematopoietic stem cell marker c-kit and (O) a chondrocyte marker Sox9.